ATP-sensitive potassium (KATP) channels couple cell metabolism to membrane excitability and are important for regulating hormone secretion, neuronal activity, and adaptive response to ischemic injuries. Dysfunction of KATP channels underlies several human diseases, referred to as KATP channelopathies. Our long-term goal is to understand the structure-function relationships of KATP channels in health and disease in order to develop mechanistic therapies for KATP channelopathies. KATP channels are unique in requiring co-assembly of an inwardly rectifying K+ channel Kir6 with a sulfonylurea receptor (SUR), an ABC transporter, for functional expression. In pancreatic ?-cells, channels consisting of Kir6.2 and SUR1 regulate insulin secretion. Mutations that reduce channel function cause congenital hyperinsulinism (CHI), whereas those that increase channel function cause neonatal diabetes and DEND syndrome. Our work in the previous funding cycles analyzing disease mutations and novel channel modulators has revealed the importance of subunit interactions in channel biogenesis and gating regulation. The finding forms the basis of our current hypothesis that SUR1 has evolved to assemble with and regulate the function of Kir6.2 via unique structural contacts, and that these molecular interfaces are modulated by physiological and pharmacological ligands to regulate channel biogenesis and gating. To test this hypothesis and to harness new pharmacological ligands for disease treatment, the following three specific aims are proposed: (1) Determine high-resolution KATP channel structures in ligand-free and ligand-bound states by single-particle negative stain- and cryo-electron microscopy (EM). (2) Determine ligand-induced structural changes in KATP channels using site-directed, unnatural amino acid- mediated crosslinking (XL) as well as a protein footprinting approach that employs chemical XL coupled with mass spectrometry (MS) analysis. (3) Exploit new pharmacological ligands we recently identified or we propose to identify through chemical library screening for correcting molecular defects caused by KATP channel mutations. The proposal is innovative in interweaving state-of-the-art single-particle EM, unnatural amino acid mediated XL, MS-based protein footprinting, and electrophysiology to gain a holistic understanding of the structural mechanisms underlying channel biogenesis and gating. The research is highly significant with regard to both human health and basic science. It is expected to generate transformative high-resolution channel structural information to advance the field and to facilitate future drug design thus opening new therapeutic opportunities for several devastating rare diseases caused by KATP mutations and also potentially for common diseases such as type II diabetes. Moreover, the research will shed light on the interesting question of how a silent ABC transporter and an ion channel have co-evolved structurally to interact with each other for metabolic regulation. Such knowledge is expected to have broader impact on our understanding of other KATP channels critical for cardiovascular, muscle and neuronal functions, and even other ABC transporters and ion channels.